Background: The glaciation cycles that occurred throughout the Pleistocene in western North America caused frequent shifts in species’ ranges with important implications for models of species divergence. For example, long periods of allopatry during species’ range contractions allowed for the accumulation of differences between separated populations promoting lineage divergence. In contrast, range expansions during interglacial periods may have had homogenizing effects via increased gene flow following secondary contact. These range dynamics are particularly pronounced in the Sierra Nevada, California, given the complex topography and climatic history of the area, thus providing a natural laboratory to examine evolutionary processes that have led to the diversity patterns observed today. Results: Here we examined the role of late Pleistocene climate fluctuations on the divergence of the Sierra Nevada endemic Alpine Chipmunk (Tamias alpinus) from its sister taxon, western populations of the Least Chipmunk (T. minimus) from the Great Basin. We used one mitochondrial gene (cytochrome b) and 14 microsatellite loci to examine the evolutionary relationship between these species. Mitochondrial sequence data revealed that T. alpinus and T. minimus populations share mitochondrial haplotypes with no overall geneaological separation, and that diversity at this locus is better explained by geography than by species’ boundaries. In contrast, the microsatellite analysis showed that populations of the same species are more similar to each other than they are to members of the other species. Similarly, a morphological analysis of voucher specimens confirmed known differences in morphological characters between species providing no evidence of recent hybridization. Coalescent analysis of the divergence history indicated a late Pleistocene splitting time (~450 ka) and subsequent, though limited, gene flow between the two lineages. Conclusions: Our results suggest that the two species are distinct and there is no contemporary introgression along their geographic boundary. The divergence of T. alpinus during this time period provides additional evidence that Pleistocene glacial cycles played an important role in diversification of species in Sierra Nevada and North America in general.